The present invention relates to phytase, nucleic acids encoding phytase, as well as the production of phytase and its use.
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Phosphorous (P) is an essential element for growth. A substantial amount of the phosphorous found in conventional livestock feed, e.g., cereal grains, oil seed meal, and by products that originate from seeds, is in the form of phosphate which is covalently bound in a molecule know as phytate (myo-inositol hexakisphosphate). The bioavailability of phosphorus in this form is generally quite low for non-ruminants, such as poultry and swine, because they lack digestive enzymes for separating phosphorus from the phytate molecule.
Several important consequences of the inability of non-ruminants to utilize phytate may be noted. For example, expense is incurred when inorganic phosphorus (e.g., dicalcium phosphate, defluorinated phosphate) or animal products (e.g., meat and bone meal, fish meal) are added to meet the animals"" nutritional requirements for phosphorus. Additonally, phytate can bind or chelate a number of minerals (e.g., calcium, zinc, iron, magnesium, copper) in the gastrointestinal tract, thereby rendering them unavailable for absorption. Still further, most of the phytate present in feed passes through the gastrointestinal tract, elevating the amount of phorphorous in the manure. This leads to an increased ecological phosphorous burden on the environment.
Ruminants, such as cattle, in contrast, readily utilize phytate thanks to an enzyme produced by rumen microorganisms known as phytase. Phytase catalyzes the hydrolysis of phytate to (1) myo-inositol and/or (2) mono-, di-, tri-, tetra- and/or penta-phosphates thereof and (3) inorganic phosphate. Two different types of phytases are known: (1) a so-called 3-phytase (myo-inositol hexaphosphate 3-phosphohydrolase, EC 3.1.3.8) and (2) a so-called 6-phytase (myo-inositol hexaphosphate 6-phosphohydrolase, EC 3.1.3.26). The 3-phytase hydrolyses first the ester bond at the 3-position, whereas the 6-phytase hydrolyzes first the ester bond at the 6-position.
Microbial phytase, as a feed additive, has been found to improve the bioavailability of phytate phosphorous in typical non-ruminant diets (See, e.g., Cromwell, et al, 1993). The result is a decreased need to add inorganic phosphorous to animal feeds, as well as lower phosphorous levels in the excreted manure (See, e.g., Kornegay, et al, 1996).
Despite such advantages, few of the known phytases have gained widespread acceptance in the feed industry. The reasons for this vary from enzyme to enzyme. Typical concerns relate to high manufacture costs, and/or poor stability/activity of the enzyme in the environment of the desired application (e.g., the pH/temperature encountered in the processing of feedstuffs, or in the digestive tracts of animals).
It is, thus, generally desirable to discover and develop novel enzymes having good stability and phytase activity for use in connection with animal feed, and to apply advancements in fermentation technology to the production of such enzymes in order to make them commercially viable. It is also desirable to ascertain nucleotide sequences which can be used to produce more efficient genetically engineered organisms capable of expressing such phytases in quantities suitable for industrial production. It is still further desirable to develop a phytase expression system via genetic engineering which will enable the purification and utilization of working quantities of relatively pure enzyme.
The present invention provides for a purified enzyme having phytase activity which is derived from a microbial source, and preferably from a fungal source, such as a Penicillium species, e.g., P. hordei (formerly P. hirsutum; ATCC No. 22053), P. piceum (ATCC No. 10519), or P. brevi-compactum (ATCC No. 48944).
The present invention further provides a polynucleotide sequence coding for the enzyme comprising a DNA as shown in any one of FIGS. 1A-1C; a polynucleotide which encodes the amino acid sequence shown in FIG. 2; a polynucleotide which encodes a phytase which comprises an amino acid segment which differs from the sequence in FIG. 2, provided that the polynucleotide encodes a derivative of the phytase specifically described herein; and a polynucleotide which encodes a phytase that comprises an amino acid segment which differs from the sequence in FIG. 2, provided that the polynucleotide hybridizes under medium to high stringency conditions with a DNA comprising all or part of the DNA in any one of FIGS. 1A-1C (SEQ ID NOs 1-3).
The present invention also provides a polynucleotide encoding an enzyme having phytate hydrolyzing activity and including a nucleotide sequence as shown in FIG. 17 (SEQ ID NO:23); a polynucleotide which encodes the amino acid sequence shown in FIG. 17 (SEQ ID NO:24); a polynucleotide which encodes a phytase which comprises an amino acid segment which differs from the sequence in FIG. 17 (SEQ ID NO:23), provided that the polynucleotide encodes a derivative of the phytase specifically described herein; and a polynucleotide which encodes a phytase that comprises an amino acid segment which differs from the sequence in FIG. 17 (SEQ ID NO:24), provided that the polynucleotide hybridizes under medium to high stringency conditions with a nucleotide sequence as shown in FIG. 17 (SEQ ID NO:23).
Additionally, the present invention encompasses vectors which include the polynucleotide sequences described above, host cells which have been transformed with such polynucleotide or vectors, fermentation broths comprising such host cells and phytase proteins encoded by such polynucleotide which are expressed by the host cells. Preferably, the polynucleotide of the invention is in purified or isolated form and is used to prepare a transformed host cell capable of producing the encoded protein product thereof. Additionally, polypeptides which are the expression product of the polynucleotide sequences described above are within the scope of the present invention.
In one embodiment, the present invention provides an isolated or purified polynucleotide derived from a fungal source of the genus Penicillium, which polynucleotide comprises a nucleotide sequence encoding an enzyme having phytase activity. The fungal source can be selected, for example, from the group consisting of Penicillium piceum and Penicillium hordei.
According to one embodiment, the polynucleotide encodes an phytate-hydrolyzing enzyme including an amino acid sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identity to an amino acid sequence as disclosed in SEQ ID NO: 4.
One embodiment of the present invention provides an isolated polynucleotide comprising a nucleotide sequence (i) having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identity to a nucleotide sequence as disclosed in SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3, or (ii) being capable of hybridizing to a probe derived from the nucleotide sequence disclosed in SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3 under conditions of intermediate to high stringency, or (iii) being complementary to the nucleotide sequence disclosed in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.
Another aspect of the present invention provides an isolated polynucleotide encoding an enzyme having phytase activity, wherein the enzyme is derived from a Penicillium source. The source can be selected, for example, from the group consisting of Penicillium piceum and Penicillium hordei. 
In one embodiment, the polynucleotide encodes a phytate-hydrolyzing enzyme that includes an amino acid sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identity to an amino acid sequence as disclosed in SEQ ID NO: 4.
In another embodiment, the polynucleotide has at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identity to a nucleotide sequence as disclosed in SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3, or (ii) is capable of hybridizing to a probe derived from the nucleotide sequence disclosed in SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3 under conditions of medium to high stringency, or (iii) is complementary to the nucleotide sequence disclosed in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.
Yet a further aspect of the present invention provides an expression construct including a polynucleotide sequence (i) having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identity to a nucleotide sequence as disclosed in SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3, or (ii) being capable of hybridizing to a probe derived from the nucleotide sequence disclosed in SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3 under conditions of medium to high stringency, or (iii) being complementary to the nucleotide sequence disclosed in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. Also provided are a vector (e.g., a plasmid) including such expression construct, and a host cell (such as an Aspergillus, e.g., Aspergillus niger or Aspergillus nidulans) transformed with such a vector.
In another of its aspects, the present invention provides a probe for use in detecting nucleic acid sequences coding for an enzyme having phytase activity derived from a microbial source, comprising: a nucleotide sequence (i) having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identity to a nucleotide sequence as disclosed in SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3, or (ii) being capable of hybridizing to a polynucleotide including a sequence as disclosed in SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3 under conditions of medium to high stringency, or (iii) being complementary to the nucleotide sequence disclosed in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.
In one embodiment, the microbial source is a fungal source, e.g., a Penicillium species, such as Penicillium hordei or Penicillium piceum. 
The present invention additionally provides a food or animal feed including an enzyme having phytase activity, wherein the enzyme comprises an amino acid sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identity to an amino acid sequence as disclosed in SEQ ID NO: 4.
The present invention provides food or animal feed including an enzyme having phytase activity, wherein the enzyme is derived from a fungal source selected from the group consisting of Penicillium hordei and Penicillium piceum. 
One aspect of the present invention provides an isolated phytase enzyme wherein the enzyme is obtained from a fungus selected from the group consisting of P. piceum and P. hordei, and has the following physiochemical properties: (1) Molecular weight: between about 45-55 kDa (non-glycosylated); and (2) Specificity: phytate.
In one embodiment, the present invention provides an enzyme derived from a fungal species (e.g., a Penicillium, such as P. piceum and P. hordei), or encoded by a nucleotide sequence capable of hybridising to SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, or the polynucleotide sequence of FIG. 17 (SEQ ID NO:23) under conditions of intermediate to high stringency, and having one or more of the following physiochemical properties:
(1) Molecular weight: between about 45-60 kDa (non-glycosylated) [based on a protein of 489 amino acids];
(2) An activity that is specific towards phytate, phytic acid or myo-inositol hexaphosphate, and/or lower phosphate derivatives thereof;
(3) A theoretical pI of between about 7 and 7.6; e.g., 7.3;
(4) A pH optimum within a range of about 4.5-5.5, e.g., about 5; and/or
(5) An ambient temperature optimum of 40-45 degree C., e.g, 42-44 degree C.
Another aspect of the present invention provides a method of producing an enzyme having phytase activity, comprising:
(a) providing a host cell transformed with an expression vector comprising a polynucleotide as described herein;
(b) cultivating the transformed host cell under conditions suitable for the host cell to produce the phytase; and
(c) recovering the phytase.
According to one embodiment, the host cell is an Aspergillus species, such as A. niger or A. nidulans. 
In another of its aspects, the present invention provides a method of separating phosphorous from phytate, comprising the step of treating the phytate with an enzyme comprising an amino acid sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identity to an amino acid sequence as disclosed in SEQ ID NO: 4.
The present invention further provides a method of separating phosphorous from phytate, comprising the step of treating the phytate with an enzyme as defined above.
Another aspect of the present invention provides a phytate-hydrolyzing enzyme that includes an amino acid sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identity to an amino acid sequence as disclosed in FIG. 17.
A further aspect of the present invention provides an isolated polynucleotide including a nucleotide sequence (i) having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identity to a nucleotide sequence as disclosed in FIG. 17, or (ii) being capable of hybridizing to a probe derived from the nucleotide sequence disclosed in FIG. 17 (SEQ ID NO:23) under conditions of intermediate to high stringency, or (iii) being complementary to the nucleotide sequence disclosed in FIG. 17 (SEQ ID NO:23).
In one embodiment, the isolated polynucleotide encodes a phytate-hydrolyzing enzyme derived from Penicillium piceum or Penicillium hordei. The enzyme includes, according to one embodiment, an amino acid sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identity to an amino acid sequence as disclosed in FIG. 17.
In another embodiment, the polynucleotide includes a nucleotide sequence (i) having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identity to a nucleotide sequence as disclosed in FIG. 17 (SEQ ID NO:23), or (ii) capable of hybridizing to a probe derived from the nucleotide sequence disclosed in FIG. 17 (SEQ ID NO:23) under conditions of medium to high stringency, or (iii) complementary to the nucleotide sequence disclosed in FIG. 17 (SEQ ID NO:23).
Another aspect of the present invention provides an expression construct comprising a polynucleotide including a nucleotide sequence (i) having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identity to a nucleotide sequence as disclosed in FIG. 17 (SEQ ID NO:23), or (ii) being capable of hybridizing to a probe derived from the nucleotide sequence disclosed in FIG. 17 (SEQ ID NO:23) under conditions of medium to high stringency, or (iii) being complementary to the nucleotide sequence disclosed in FIG. 17 (SEQ ID NO:23). The present invention further provides a vector (e.g., plasmid) including such an expression construct, as well as a host cell (e.g., Aspergillus niger or Aspergillus nigulans) transformed with such a vector.
The present invention additionally provides a probe for use in detecting nucleic acid sequences coding for an enzyme having phytase activity derived from a microbial source, comprising: a nucleotide sequence (i) having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identity to a nucleotide sequence as disclosed in FIG. 17 (SEQ ID NO:23), or (ii) being capable of hybridizing to a polynucleotide including a sequence as disclosed in FIG. 17 (SEQ ID NO:23) under conditions of medium to high stringency, or (iii) being complementary to the nucleotide sequence disclosed in FIG. 17 (SEQ ID NO:23).
In one embodiment, the microbial source is a fungal source, e.g., a Penicillium species, such as P. hordei or P. piceum. 
The present invention further provides a food or animal feed including an enzyme having phytase activity, wherein the enzyme includes an amino acid sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identity to an amino acid sequence as disclosed in FIG. 17 (SEQ ID NO:24).
Still further, the present invention provides a method of separating phosphorous from phytate, comprising the step of treating the phytate with an enzyme (i) having phytate hydrolyzing activity and (ii) including an amino acid sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identity to an amino acid sequence as disclosed in FIG. 17 (SEQ ID NO:24).
As will be appreciated, an advantage of the present invention is that a polynucleotide has been isolated which provides the capability of isolating further polynucleotides which encode proteins having phytase activity.
Another advantage of the present invention is that, by virtue of providing a polynucleotide encoding a protein having phytase activity, it is possible to produce through recombinant means a host cell which is capable of producing the protein having phytase activity in relatively large quantities.
Yet another advantage of the present invention is that commercial application of proteins having phytase activity is made practical. For example, the present invention provides animal feed incorporating the phytase described herein.
Still a further advantage of the present invention is that it provides an enzyme having phytate hydrolyzing activity, with such activity being optimum at temperatures from about 40 to about 45 degrees C, which make it very suitable to for use in animal feed (i.e., the enzyme has high activity at the point of action, in the gut of an animal).
Other objects and advantages of the present invention will become apparent from the following detailed specification.